by the institution veterinarian (Dr. Gregory Heisey). The cost for this is incorporated into the per diem costs. It is anticipated that the core will prepare 54 dogs per year. Of these 42 will be allocated to Project 2 and 12 to Project 1. In addition, the core will budget for the purchase of an additional 15 dogs, if necessary, to account for early mortality from coronary microembolizations which currently stands at ~15%. Specific Aim 2: To execute pharmacologic protocols involving coronary microembolization-induced HF i.e. assume responsibility for treatment regimens. Due to the extensive experience of the laboratory of Dr. Sabbah with the canine coronary microembolization-induced HF model, centralizing the location of these procedures would ensure the consistency of the model and reduce the incidence of technical errors. Since the Core will be housing the dogs for these experiments, it is logical that the Core should also oversee the pharmacologic treatment of these animals. Randomization and drug treatment will be supervised by Drs. Sabbah and Sharov and managed on a daily basis by Ms. Vich and Ms. Hazel both of who have at least 10 years experience in pharmacologic treatment of dogs with heart failure as members of Dr. Sabbah's laboratory staff. Treatment doses and time of day administered will be recorded in a daily log initialed by the person administering the agent. Each dog file will contain all information pertaining to the dog'shealth,handlingandtreatmentregimens.TheCoreleader will periodically review these files to ensure that each dog is receiving the proper treatment and will likely survive the study. The canine model of chronic HF used in the present study has been previously described in detail (1). Chronic LV dysfunction and failure will be produced by multiple sequential intracoronary embolizations with polystyrene Latex microspheres (70-102 _ma in diameter), which results in loss of viable myocardium. Embolizations will be performed 1 to 3 weeks apart and will be discontinued when LV ejection fraction, determined angiographicaUy, is < 30 %. Microembolizations will be performed during cardiac catheterization under general anesthesia and sterile conditions. The anesthesia regimen will consist of a combination of an intravenous injection of oxymorphone (0.22 mg/kg), diazepam (0.17 mg/kg), and sodium pentobarbital (150 - 250 mg to effect). This protocol is approved by the Henry Ford Health System Institutional Animal Care and Use Committee and conforms to the "Position of the American Heart Association on Research Animal Use" and the Guiding Principles of the American Physiological Society. Angiographic Measurements Left ventriculograms will be obtained prior to each microembolization session to determine the extent of LV dysfunction with the dog placed on its right side. Ventriculograms will be recorded on 35 mm cine at 30 frames per second during the injection of 20 ml of contrast material (RENO-M-60, Squibb). Correction for image magnification will be made with a calibrated radiopaque grid placed at the level of the LV. LV ejection fraction will be calculated as the ratio of the difference between end-diastolic and end-systolic volumes divided by end-diastolic volume times 100. Extrasystolic and post- extrasystolic beats will be excluded from the angiographic analysis. Regional LV wall motion using both the ventriculogram and 2-dimensional echocardiograms will be used to select the coronary artery (left circumflex coronary artery or left anterior descending coronary artery) into which the 2nd, 3rd, 4th etc. microembolizations will be administered. This procedure of weekly coronary microembolizations will be followed until the target LV ejection fraction is reached. We anticipate based on years of experience that an average of 7 microembolization sessions will be needed for each dog to achieve the target LV ejection fraction. Once a the target ejection fraction is reached, the dog will be maintained for 2 weeks before being randomized into studies within Project 2 or shipped to Cleveland for use in Project 1. The period of 2 weeks is needed to ensure that microinfarcts produced by the last microembolization are fully healed. Assignment of Animals to Project I and 2. For Project 1, dogs will be shipped to Cleveland by Hodgin's Kennels once the 2 weeks post last embolization have elapsed. Dr. Sabbah's laboratory has been using Hodgin's Kennels to ship dogs for 4 years and has been pleased with their services. The dogs will be transported in air-conditioned vans containing individual cages. Dogs for Project 2 will remain in Detroit and released from the Core to the Project for one day prior to microembolizations for baseline measurements. The dogs will then be returned to the Core for induction of HF by coronary microembolization. After the target ejection fraction has been reached, the dogs will be released again to Project 2, for one day, for pre-treatment measurements. The dogs will then return to the Core for daily care and treatment until 6, 12 or 16 weeks have passed, depending on which schedule the dog has been randomized to. Once the appropriate time period has been reached, the dogs will be released to the Project for the final measurements and then returned to the Core. The Core will arrange for shipment of the dogs to Cleveland for use by Projects 1 and 4 and for use of tissue by the Metabolism Core. 2.50 Specific Aim 3: To coordinate distribution of dogs and tissues derived from coronary microembolization-induced HF experiments. The Core will coordinate delivery of dogs to Projects 1, 2 and 4. Dogs will be shipped to Cleveland for Project 1 by Hodgin's Kennels. Samples will be processed by the Metabolism Core and shipped back to Detroit overnight on dry ice. Once samples have arrived in Detroit, they will be processed for histology to meet the needs of Project 2. Frozen tissue samples will also be given to Project 2 for biochemical analysis. Tissue from Project 3 will be shipped to the Core for histological evaluation. These shipments will be handled between the Core Leader (Dr. Sabbah) and the Project 3 Leader (Dr. Recchia) to ensure that the tissue is properly processed and delivered in a timely manner. Specific Aim 4: To perform histological analysis of tissue samples derived from HF dogs. Tissue samples for histological analysis will be obtained from coronary microembolization-induced HF dogs as well as dogs undergoing rapid ventricular pacing (see Project 3). The Core will analyze tissue from both models. The Core Co-Leader (Dr. Sharov) will oversee the processing and microscopic analysis of samples, as well as acquisition of supplies for histological analysis. Dr. Sharov will also ensure proper maintenance of the equipment involved in these studies. Once all in-vivo measurements are completed in Cleveland and New York (Projects 1 and 3, respectively), the heart will be rapidly removed and placed in ice-cold cardioplegia solution. From each heart, 3 transverse slices one from the basal third, middle third and apical third of the LV, each approximately 3 mm thick, will be obtained. From each transverse slice transmural tissue blocks will be processed and shipped to Detroit (location of the Core). Similarly, LV free wall tissue blocks will be obtained from a second mid-ventricular transverse slice, mounted on cork using Tissue-Tek embedding medium and rapidly frozen in isopentane pre-cooled in liquid nitrogen and stored at -70[unreadable]C until shipped to Detroit overnight on dry ice. In Detroit, tissue blocks will be embedded in formalin. From each block, 6 lain thick sections will be prepared and stained with Gomori trichrome to identify fibrous tissue. The volume fraction of replacement fibrosis namely, the proportion of scar tissue to viable tissue in all three transverse LV slices, will be calculated as the percent total surface area occupied by fibrous tissue using computer-based video densitometry (MOCHA, Jandel Scientific). Cryostat sections, approximately 8 Jam thick, will be prepared from each liquid nitrogen frozen blocks and stained with fluorescein-labeled peanut agglutinin after pretreatment with 3.3 U/ml neuroaminidase type V (Sigma Chemical Co.) to delineate the myocyte border and the interstitial space including capillaries. Sections will be double stained with rhodamine-labeled Griffonia simplicifolia lectin I (GSL-I) to identify capillaries. Ten radially oriented microscopic fields (magnification X100, objective X40, and ocular 2.5) will be selected at random from each section for analysis. Fields containing scar tissue (infarcts) will be excluded. An average myocyte cross-sectional area will be calculated for each dog using computer-assisted planimetry. The total surface area occupied by interstitial space and the total surface are occupied by capillaries will be measured from each randomly selected field using computer-based video densitometry (MOCHA, Jandel Scientific). The volume fraction of interstitial collagen will be calculated as the percent total surface area occupied by interstitial space minus the percent total area occupied by capillaries. Capillary density will be calculated as the number of capillaries per mm 2. Oxygen diffusion distance will be calculated as [unreadable] the distance between two adjoining capillaries. For comparison, measurements ofmyocyte cross-sectional area and volume fraction of interstitial fibrosis will be made using identical techniques in LV tissue sections obtained from 7 normal dogs. 251 5. Description of Facilities The Cardiovascular Research Laboratories are located on the 4 th floor of the Henry Ford Hospital Education and Research Building. The 7 story facility was built in 1976 and is part of the Hem'y Ford Hopsital complex. It houses active research laboratories of various medical disciplines, office space, and research animal quarters. The Cardiovascular Research Laboratories consist of a total of 4000 square feet of research space. The laboratory floor plan is shown below. Several laboratories exist within Cardiovascular Research and are under the direct supervision of the Director (Dr. Hani N. Sabbah). The day-to-day management of the laboratories is the responsibility of laboratory group leaders that are Senior Staff Investigators. The following laboratories constitute Cardiovascular Research: Physiology (hemodynamics and echocardiography); biochemistry and molecular biology; cell electrophysiology and single cell function; and histopathology (includes immunohistochemistry, histomorphometry and transmission electron microscopy). Cardiovascular Research Laboratories Layout 14 1: 19 z 7 10 l 5 11 12 13 1. 35 mm Film Processor 2. Office (Senior Stafflnvestigator) 3. Office Secretary to Dr. Sabbah 4. Director's Office (Dr. H.N. Sabbah) 5. Office (Senior Staff Investigator) 6. Biochemistry & Molecular Biology Laboratory 7. Cell Electrophysiology Laboratory 8. Biochemistry & Molecular Biology Laboratory 9. Cold Room 10. Biochemistry Laboratory 11. Animal Catheterization laboratory #1(Seimens) 12. Pathology / Histology Laboratory 13. Histomorphometry Laboratory 14. Organ Bath Physiology Laboratory 15. Office (Dr. Sharov) 16. Office Drs. Tanhehco and Todor 17. Office (Dr. Morita and Research Fellows) 18. Animal Catheterization Laboratory #2 (General Electric)